TNF-? is an inflammatory cytokine that affects multiple cells types in numerous inflammatory diseases. In asthma, TNF-? mediates inflammation through its action on cells that traffic into the lung and on resident lung cells, including airway smooth muscle (ASM) cells. Unfortunately, therapeutic targeting of TNF-? using anti-TNF-? antibodies has been disappointing due to safety concerns. Herein we propose to elucidate novel signaling pathways transduced by TNF-? via the scaffold protein TNFR1-Ubiquitous Scaffolding and Signaling Protein (TRUSS). We will extend previous limited analysis of TRUSS restricted to transformed cells to identify new signaling interactions and pathways in physiologically relevant cells, primary ASM cells. Importantly, we will also link these signaling events to regulation of expression of genes important in airway inflammation in the lung, thus identifying new mechanisms and targets through which ASM functions as an immunomodulatory cell. Our goal is to explore novel components of the TNF-? signaling pathways that may offer safer alternative therapeutic options for anti-TNF-? therapy. To this end, two aims in ASM cells will be explored: determine the unique contribution of endogenous TRUSS to the regulation of NF-kB activation (Aim 1) and ii) evaluate the selective contribution of endogenous TRUSS to the regulation of NF-kB-associated genes (Aim 2). In both aims, siRNA, mutated constructs and expression vectors will be used to modulate the expression of TRUSS, after which NF-kB-mediated transactivation activities, NF-kB phosphorylation and acetylation, in vitro kinase assays to assess IKK activity, phosphorylation of IKK?/?, NF-kB-DNA interactions, and the transcriptional regulation of NF-kB-dependent genes will be analyzed using state-of-the-art molecular and biochemical approaches already established in our laboratories. This comprehensive molecular approach is expected to expand our understanding of how TNF-? mechanistically regulates NF-kB-associated genes in physiologically relevant cells. Elucidating TRUSS-dependent signaling events opens up the opportunity for more selective targeting of TNF-driven events. Findings from these studies will provide new insight into fundamental TNF biochemistry, and provide an empirical basis to explore the role of TRUSS in more integrative disease model, with the ultimate goal of developing (safer) therapeutics that target specific TNF-? signals critical to inflammatory disease pathogenesis.